This application is a 371 of PCT/EP00/07490 filed Aug. 2, 2000.
The present invention relates to 32P- or 33P-labelled bisphosphonates as radiotherapeutic radiopharmaceuticals. The 32P or 33P-labelled bisphosphonates, which are chemically identical to the unlabelled agent, are expected to target the lesion site in an identical manner, but also deliver a significant radiocytotoxic effect to the surrounding cells. This should result, given the favourable energetics of the xcex2 particle emission from the 32P nuclide, in a loss of proliferative capacity of cells associated with the tumour lesion. The relative stability and in vivo localisation of bisphosphonates makes them good candidates as 32P/33P delivery vehicles.
Bisphosphonates are known as palliatives to treat osteosarcoma or bone metastases associated with carcinoma such as breast or prostate. These agents, such as Pamidronate and Clodronate, exert a negative effect upon osteoclasts at the site of the lesion resulting in decreased bone resorption at this site. Bisphosphonates, however, appear to lose activity with time necessitating repeat administration.
There have been extensive examples of bisphosphonate syntheses in the literature over the last 25 years. Synthesis of the xcex1-hydroxyl-methylene bisphosphonates, those most commonly studied with respect to bone disorders, have largely been performed under harsh conditions of elevated temperatures, often resulting in low-yielding reactions. The common route of synthesis to such compounds involves heating a source of phosphorus, usually phosphorous acid, with the appropriate carboxylic acid and PCI3. This synthesis is not ideal for the incorporation of 32/33P into a bisphosphonate, due to the safety and radiological hazards associated with volatile 32/33PCI3, and the fact that the primary role of the PCI3 is to generate an activated carbonyl compound rather than as a source of the phosphonate groups. The poor yields often obtained from the traditional syntheses are not appropriate for radiolabelling with an expensive radionuclide.
Tris(trimethylsilyl) phosphite, P(OTms)3, has been isynthesised from PCI3 and, alternatively, from phosphorous acid and, subsequently, used to introduce phosphorus into compounds.
U.S. Pat. No. 3,965,254 describes the use of 32/33P in bisphosphonates for the treatment of bone cancer. It was shown that the 32P or 33P radionuclide could be targeted to the tumour site. The patent describes the incorporation of 32P into EHDP (disodium ethane-1-hydroxy-1,1-diphosphonate) and its subsequent use in in vivo studies. The synthesis used to generate the radiolabelled bisphosphonates followed the conventional synthesis using 32/33PCI3. Within the synthetic route the reaction is, at times heated to 145xc2x0 C. for up to 6 hours and a reflux of 40 hours duration. The final yield was approximately 65%. This route of preparation for these compounds is seen as inappropriate for the reasons outlined above.
Accordingly, it is one object of this invention to supply a convenient and improved route of synthesis for radiolabelled bisphosphonates, for the use in therapeutic treatment of bone metastatic disease.
In one aspect the invention provides a method of making a bisphosphonate, which method comprises reacting a tris(silyl)phosphite with an activated carbonyl compound and hydrolysing the resulting intermediate according to the reaction scheme:
xe2x80x83P(OX)3+RCO.Yxe2x86x92Intermediatexe2x86x92RC(OH)(PO.[OH]2)2
where
each X is the same or different and is tri-(C1-C12 hydrocarbyl)silyl,
R is C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C2-C12 aryl (including heteroaryl) or substituted variants of these where functionalised groups, if present, are appropriately masked (ie. protected) during the synthesis,
and Y is an activating moiety.
The functional group(s) of the xe2x80x98substituted variantsxe2x80x99 can be amino (primary, secondary or tertiary), hydroxy, alkoxy or fluorine. R is preferably C1-C12 alkyl, C1-C12 fluoroalkyl or C1-C12 primary, secondary or tertiary aminoalkyl or a derivative of these, or a substituted alkyl group containing nitrogen as part of a heterocyclic ring system. R is most preferably C1-C6 alkyl or C2-C9 primary, secondary or tertiary aminoalkyl. Preferred C2-C9 aminoalkyl groups are xe2x80x94(CH2)pNQ2 where p is 2 or 3 and Q is H or C1-C5 alkyl, with xe2x80x94(CH2)2NH2, xe2x80x94(CH2)3NH2 and (CH2)3NMe(pentyl) being especially preferred.
In the starting tris(silyl)phosphite P(OX)3, X is tri-(C1-C12 hydrocarbyl)silyl, e.g. trialkylsilyl or triarylsilyl, conveniently trimethylsilyl since derivatised trimethylsilanes are readily commercially available. Mixed phosphites are possible and may be preferred.
The starting activated carbonyl compound of formula RCO.Y is preferably an acid halide, particularly an acid chloride or acid bromide; an acid anhydride; an xcex1-ketophosphonate; or an active ester such as that derived from N-hydroxysuccinimide. Hence the activating moiety Y can be: a leaving group such as halogen (especially Cl or Br), or an acid anhydride linkage (RCO)2O, or an active ester, examples of which are well known to those skilled in the art. Alternatively, Y can be a phosphonate xe2x80x94PO(ORxe2x80x2xe2x80x3)2, ie. RCOY may be an xcex1-ketophosphonate. Preparation of bisphosphonates has also been achieved using an active ester derived from 2-hydroxypyridine and also acid anhydrides. Active esters derived from pentafluorophenol and hydroxybenztriazole are also possible. These reactions show how the increased nucleophilicity of the silylated phosphites (compared with trialkyl phosphites) permits the use of much less activated carbonyl compounds in the formation of the desired products.
When Y is a leaving group, the Intermediate generally has the formula RC(OZ)(PO.[OX]2)2 where Z is H or X. When RCOY is an xcex1-ketophosphonate, an addition reaction rather than a substitution reaction may take place, e.g.:
P(OX)3+RCO.PO(ORxe2x80x2xe2x80x3)2xe2x86x92RC(OZ)(PO)[OX]2)(PO[ORxe2x80x2xe2x80x3]2)xe2x86x92RC(OH)(PO[OH]2)2
where Rxe2x80x2xe2x80x3 is C1-12 alkyl or X (by treatment of the xe2x80x94O-alkyl system with TmsBr or Tmsl).
The R group is chosen to provide a desired substituent on the hydroxymethane-bisphosphonate unit.
Various bisphosphonate drugs R1CR2(PO[OH]2)2 have been commercialised as follows:
Preferably the tris(silyl)phosphite contains 32P or 33P, either at 100% abundance or at least at an artificially high isotopic abundance of e.g. at least 1%. Preferably 2 molar equivalents of the tris(silyl)phosphite are reacted with 1 molar equivalent of the activated carbonyl compound. Although use of elevated temperatures is possible, the reaction is found to go in high yield at ambient temperature, thus providing a convenient route for introducing 32P or 33P into a bisphosphonate molecule of choice.
Dry aprotic solvents such as diethyl ether or tetrahydrofuran can be used to help facilitate handling of the reactants. Reaction times depend on the leaving group and on the quantity of solvent used. With modest quantities of solvent, reaction times are typically 10 to 15 min with an acid chloride or anhydride but it can require heating for several hours with less activated carbonyl compounds.
There results an intermediate having the formula RC(OZ)(PO.[OX]2)2 or RC(OZ)(PO)[OX]2)(PO[ORxe2x80x2xe2x80x3]). In general these intermediates are believed novel and form further aspects of this invention. They may readily be hydrolysed to the desired bisphosphonates by the addition of excess methanol or water at elevated or preferably ambient temperature. Some of the resulting bisphosphonates are new compounds.
Thus the invention also provides radiolabelled bisphosphonates of the formula:
Rxe2x80x3C(OH)(nPO[OH]2)2
where
n is at least partly 32 or 33 whereby the compound contains an artificially high proportion of 32P or 33P,
and Rxe2x80x3 is C1-C12 primary, secondary or tertiary aminoalkyl or a derivative of these, or a substituted alkyl group containing nitrogen as part of a heterocyclic ring system. Rxe2x80x3 is preferably C2-C9 primary, secondary or tertiary aminoalkyl, most preferably xe2x80x94(CH2)pNQ2 where p is 2 or 3 and Q is H or C1-C5 alkyl, with xe2x80x94(CH2)2NH2, xe2x80x94(CH2)3NH2 and (CH2)3NMe(pentyl) being especially preferred.
It may be advantageous to provide one of the starting reagents, e.g. the tris(silyl)phosphite POX3 or the activated carbonyl compound RCOY, in immobilised form. This facilitates purification of the starting compound, which may be particularly useful when an isotopically labelled phosphite is used, and also facilitates purification of the desired bisphosphonate product.
The radiolabelled bisphosphonates of the present invention have useful properties as palliatives to treat osteosarcoma or bone metastasis associated with carcinoma such as breast or prostate, with the additional advantage of comprising an artificially high isotopic abundance of 32P or 33P for targeting either bone metastasis or tumours or other diseases so that may be treated with radiotherapy.
Also included within the scope of the invention are radiolabelled phosphorus compounds of the formula
Rxe2x80x2qnP(OX)3xe2x88x92q
where
Rxe2x80x2 is C1-C12 alkyl or C2-C12 aryl,
q is 0, 1 or 2,
n is at least partly 32 or 33 whereby the compound contains an artificially high proportion of 32P or 33P,
each X is the same or different and is tri-(C1-C12 hydrocarbyl)silyl.
When q is 0, these are radiolabelled trialkylsilylphosphites, useful as starting materials in the method of the invention described above.